Techniques for enhancing the selectivity and efficacy of antimicrobial and anticancer polymer agents

ABSTRACT

The subject disclosure is directed to techniques for enhancing the selectivity and efficacy of therapeutic polymers against a broad spectrum of pathogens and cancer cell lines. According to an embodiment, a method is provided that comprises forming a therapeutic polymer based on polymerization of a plurality of therapeutic monomers, wherein the therapeutic polymer provides a therapeutic functionality. The method further comprises attaching biotin to the therapeutic polymer, resulting in a biotin-functionalized therapeutic polymer, wherein the biotin-functionalized therapeutic polymer provides greater therapeutic efficacy relative to the therapeutic polymer.

TECHNICAL FIELD

This application generally relates to techniques for enhancing theselectivity and efficacy of therapeutic polymers against a broadspectrum of pathogens and cancer cell lines.

BACKGROUND

Nosocomial Gram negative bacteria Pseudomonas aeruginosa (P. aeruginosa)infections have high adaptability and strong antibiotics resistance. Inaddition, these bacteria have the ability to form biofilms, whichfurther increases its resistance to a broad spectrum of antibiotics. Asa result, hospital acquired P. aeruginosa infections account for highmorbidity rates. P. aeruginosa infections are also the most commonlyacquired infection among patients with cystic fibrosis and chronicobstructive pulmonary disease (COPD). Phagocytes such as neutrophils andmacrophages are responsible for eradicating intracellular bacteria viaacidic/oxidative stresses, build-up of metal in the phagolysosome,limiting its availability of key nutrients (fatty acids and iron) tophagocytosed bacteria. However, P. aeruginosa has devised mechanisms toevade the innate immune system and ingestion by macrophages. To compoundthe problem, P. aeruginosa was recently shown to survive and thrivewithin the macrophages by employing various gene mutations that preventelimination within the hostile environment during phagocytosis. Anexample, is the inhibitory effect of MgtC on adenosine triphosphate(ATP) synthase activity, enabling P. aeruginosa to withstand metabolicdysregulation during the acidification of the phagosome. In addition,the outer membrane protein of P. aeruginosa plays a critical role forits survival within the macrophage. Finally, P. aeruginosa have alsobeen found within phagocytic cells (alveolar macrophages) of infectedmice. Incomplete clearance of P. aeruginosa from these infectedphagocytic cells will ultimately lead to infection of other cell types,starting with dissemination from the original site of infection,affecting especially immunocompromised patients. Moreover, P. aeruginosawas shown to recruit extracellular deoxyribonucleic acid (DNA) andmigratory inhibition factors from neutrophils, promoting the growth ofbiofilm. The biofilm is an extracellular matrix for the bacteria toreside within, shielding the colonies from antibiotics and causingchronic infections.

To circumvent bacteria resistance and intracellular infection ofmacrophages, polyguanidines with transmembrane and highly effectiveantimicrobial properties have been employed to treat P. aeruginosainfection. Unlike traditional antibiotics and various syntheticantimicrobial polymers and antimicrobial peptides which do not enter thebacteria, the polyguanidiniums are cationic polymers which possessamphiphilicity (balance between hydrophobicity and soluble positivecharges). In this regard, the polyguanidiniums damage microbes viamembrane lysis resulting from electrostatic attraction between positivepolymer charges and negative membrane surface of microbes or viatrans-location across the membrane leading to cytosol precipitation.Currently, polymers with varying non-degradable backbones such aspolyethylenimines polyacrylates, polynorbornene, polyarylamides andmetallopolymers have been reported and studied for their antimicrobialproperties. However, the non-degradability properties of these polymerspose an issue of low selectivity and high hemotoxicity to mammaliancells. Conversely, antimicrobial peptides (degradable polypeptidebackbone) are limited in their clinical applications due tocytotoxicity, enzymatic degradation and high production cost.

In order to synthesize a low cost, well defined biodegradable syntheticantimicrobial polymer, an organocatalytic ring opening polymerization(OROP) technique has been employed to attain aliphatic polycarbonatesthat possess low toxicity and biocompatibility. In addition, monomers ofvarious functionalities can be easily incorporated into synthesis ofthese antimicrobial polycarbonates and are well characterized due to theprecise control of the OROP. This allowed for design of variousantimicrobial macromolecules, with the “same centered” design approachshown more recently to provide a distinctive combination of bothantimicrobial activity and selectivity properties. However, increasingthe hydrophobicity on the “same centered” guanidinium-functionalizedpolycarbonates did not significantly improve antimicrobial activity andreduced selectively in certain instances.

SUMMARY

The following presents a summary to provide a basic understanding of oneor more embodiments of the invention. This summary is not intended toidentify key or critical elements or delineate any scope of thedifferent embodiments or any scope of the claims. Its sole purpose is topresent concepts in a simplified form as a prelude to the more detaileddescription that is presented later. The subject disclosure relates totechniques for enhancing the selectivity and efficacy of therapeuticpolymers against a broad spectrum of pathogens and cancer cell lines.

According to an embodiment, a method is provided that comprises forminga therapeutic polymer based on polymerization of a plurality oftherapeutic monomers, wherein the therapeutic polymer provides atherapeutic functionality. The method further comprises attaching biotinto the therapeutic polymer, resulting in a biotin-functionalizedtherapeutic polymer, wherein the biotin-functionalized therapeuticpolymer provides greater therapeutic efficacy relative to thetherapeutic polymer.

In various implementations, the biotin-functionalized therapeuticpolymer provides the greater therapeutic efficacy based on increaseduptake of the biotin-functionalized therapeutic polymer by pathogens orcancer cells relative to the therapeutic polymer. In particular, thebiotin-functionalized therapeutic polymer can provide the greatertherapeutic efficacy based on reduced toxicity of the therapeuticpolymer toward mammalian cells relative to the therapeutic polymer. Inone or more implementations, the therapeutic functionality comprises ananticancer functionality. In other implementations, the therapeuticfunctionality comprises an antimicrobial functionality. In variousimplementations, the therapeutic polymer comprises polyguanidinium.

The techniques for attaching the biotin can vary. For example, in someimplementations, the attaching comprises performing the polymerizationof the plurality of therapeutic monomers in presence of biotinol,resulting in formation of the biotin-functionalized therapeutic polymerwith the biotin bound to an end of the polymer backbone. In otherimplementations, the biotin can be attached via post polymerizationmodification.

In another embodiment, a therapeutic is polymer is provided comprising apolymer backbone, therapeutic functional groups bound to the polymerbackbone, and a biotin-based functional group bound to an end of thepolymer backbone. For example, in one or more implementations thebiotin-based functional group can comprise biotinol. In variousimplementations, the therapeutic functional groups comprise guanidiniummoieties. In some implementations, the therapeutic polymer facilitatesnecrosis of bacteria cells. In other implementations, the therapeuticpolymer facilitates autophagy of cancer cells.

In some implementations, the therapeutic polymer has a chemicalstructure characterized by Formula I:

-   wherein n represents an integer between 10 and 50,-   wherein R₁ comprises the biotin-based functional group, and-   wherein R₂ comprises a spacer group.

In another implementation, the therapeutic polymer has a chemicalstructure characterized by Formula II:

-   wherein n represents an integer between 10 and 50,

In another implementation, the therapeutic polymer has a chemicalstructure characterized by Formula III:

-   wherein n represents an integer between 10 and 50,-   wherein m represents an integer between 1.0 and 10, and-   wherein R₁ comprises the biotin-based functional group.

In yet another implementation, the therapeutic polymer has a chemicalstructure characterized by Formula IV:

-   wherein n represents an integer between 10 and 50,-   wherein R₁ comprises a functional group, and-   wherein R₂ comprises the biotin-based functional group.

In one or more additional embodiments, a therapeutic polymer is providedthat has a chemical structure characterized by Formula I:

-   wherein n represents an integer between 10 and 50,-   wherein R₁ comprises the biotin-based functional group, and-   wherein R₂ comprises a spacer group.

In some implementations, the therapeutic polymer is an anticancer agent.In other implementations, the therapeutic polymer is an antimicrobialagent.

In another embodiment, an anticancer agent is described that has achemical structure characterized by Formula I:

-   wherein n represents an integer between 10 and 50, and-   where the anticancer agent is effective against a plurality of    different cancer cell lines. In one or more implementations, the    plurality of different cancer cell lines comprises cancer cell line    BT-474.

One or more additional embodiments are directed to a method thatcomprises polymerizing guanidinium-functionalized cyclic carbonatemonomers via a ring opening polymerization reaction using biotinol as aninitiator. The method further comprises forming a biotinylatedpolyguanidinium macromolecule based on the polymerizing. In variousembodiments, the biotinylated polyguanidinium provides antimicrobial andanticancer functionality.

BRIEF DESCRIPTION OF THE DRAWINGS

Numerous aspects, embodiments, objects and advantages of the presentinvention will be apparent upon consideration of the following detaileddescription, taken in conjunction with the accompanying drawings, inwhich like reference characters refer to like parts throughout, and inwhich:

FIG. 1 presents an example synthesis route 100 for generating abiotinylated polyguanidinium in accordance with various embodimentsdescribed herein.

FIG. 2 presents another example synthesis route 200 for generating abiotinylated therapeutic polymer in accordance with various embodimentsdescribed herein

FIG. 3 presents transmission electron microscopy (TEM) micrograph imagesof a cancer cell before and after treatment with a biotinylatedpolyguanidinium based anticancer agent in accordance with variousembodiments described herein.

FIGS. 4A and 4B present charts illustrating the in vitro efficacy of abiotinylated polymer and a non-biotinylated polymer against cancer cellline BT-474 (a human ductal carcinoma cell line) in accordance withvarious embodiments described herein.

FIG. 5 presents a chart illustrating the cytotoxicity of a biotinylatedpolymer against healthy non-cancerous mammalian cell line HEK293 inaccordance with various embodiments described herein.

FIG. 6 presents a high-level flow diagram of an example method forenhancing the selectivity and efficacy of therapeutic polymers against abroad spectrum of pathogens and cancer cell lines via the introductionof biotin in accordance with various embodiments described herein.

FIG. 7 presents an example synthesis route 600 for forming a therapeuticcoacervate in accordance with various embodiments described herein.

FIG. 8 provides a transmission electron microscopy (TEM) image ofComplex E in accordance with various embodiments described herein. Inthe embodiment shown,

FIG. 9A presents an example synthesis route for forming Polymer A inaccordance with various embodiments described herein. In the embodimentshown,

FIG. 9B presents an example synthesis route for forming Polymer A′ inaccordance with various embodiments described herein.

FIG. 9C presents an example synthesis route for forming Polymer J inaccordance with various embodiments described herein. In the embodimentshown,

FIG. 9D presents an example synthesis route for forming Polymer J′ inaccordance with various embodiments described herein.

FIG. 10 presents example synthesis routes for forming functionalguanidinium cationic homopolymers in accordance with various embodimentsdescribed herein.

FIG. 11 presents example synthesis routes for forming functionalguanidinium cationic copolymers in accordance with various embodimentsdescribed herein.

FIG. 12 provides a table identifying characteristics of exampletherapeutic coacervates in accordance with various embodiments describedherein.

FIGS. 13A and 13B present graphs illustrating the kinetic stabilitycharacteristics of example coacervates in accordance with variousembodiments described herein.

FIG. 14 presents a graph illustrating the in vitro release profile ofcationic polymers from some example coacervates in accordance withvarious embodiments described herein.

FIG. 15A presents a graph illustrating the antimicrobial selectivity andefficacy of example therapeutic polymers and coacervate complexesagainst Pseudomonas aeruginosa (P. aeruginosa) in accordance withvarious embodiments described herein.

FIG. 15B provides a table illustrating the antimicrobial selectivity andefficacy of example therapeutic polymers and coacervate complexesagainst P. aeruginosa in accordance with various embodiments describedherein.

FIG. 16 presents a table illustrating characteristics of examplecoacervate Complexes HJ with varying degrees of polymerization (DP) inaccordance with various embodiments described herein.

FIG. 17 presents is a graph illustrating the biocompatibility/toxicityof various therapeutic polymers and coacervate complexes with mammaliancells in accordance with various embodiments described herein

FIG. 18 provides a graph illustrating the cell viability of mammaliancells with Polymyxin B (PolyB) and a coacervate form of PolyB inaccordance with various embodiments described herein.

FIG. 19 presents a graph illustrating the intracellular P. aeruginosainfection clearance using various coacervate embodiments describedherein.

FIGS. 20A and 20B present graphs illustrating the intracellularinfection clearance of Complex E with P. aeruginosa in accordance withvarious embodiments described herein.

FIGS. 21A and 21B provide tables illustrating the in vivo toxicity andantimicrobial efficacy of coacervate complexes in accordance withvarious embodiments described herein.

FIG. 22 provides a graph demonstrating the efficacy of variouscoacervate complexes against BT-474 cancer cells in accordance withvarious embodiments described herein.

FIG. 23 provides a graph demonstrating the efficacy of variouscoacervate complexes with urea against BT-474 cancer cells in accordancewith various embodiments described herein.

FIG. 24 presents a chart illustrating the cytotoxicity of a coacervateComplex D against healthy non-cancerous mammalian cell line HEK293 inaccordance with various embodiments described herein.

FIG. 25 presents a high-level flow diagram of an example method forenhancing the selectivity and efficacy of therapeutic polymers against abroad spectrum of pathogens and cancer cell lines using a coacervatecomplex in accordance with various embodiments described herein.

FIG. 26 presents a high-level flow diagram of an example method forenhancing the selectivity and efficacy of therapeutic polymers against abroad spectrum of pathogens and cancer cell lines using a coacervatecomplex comprising a biotinylated anionic polymer in accordance withvarious embodiments described herein.

FIG. 27 provides a diagram illustrating fluorescence based coacervatediagnostics in accordance with various embodiments described herein.

FIG. 28 presents a graph illustrating the fluorescence quenchingcharacteristics of diagnostic coacervate complexes in accordance withvarious embodiments described herein.

FIG. 29 presents a high-level flow diagram of an example method fordiagnosing a disease or condition using a fluorescence based coacervateassay in accordance with various embodiments described herein.

DETAILED DESCRIPTION

The following detailed description is merely illustrative and is notintended to limit embodiments and/or application or uses of embodiments.Furthermore, there is no intention to be bound by any expressed orimplied information presented in the preceding Summary section or in theDetailed Description section.

The subject disclosure provides techniques for enhancing theantimicrobial selectivity and efficacy of aliphatic polymers, such asguanidinium-functionalized polycarbonates, against a broad spectrum ofbacterial pathogens, including P. aeruginosa. Various embodiments of thedisclosed techniques for enhancing the selectivity and efficacy ofantimicrobial polymers are further extended to anticancer blockpolymers, providing techniques for enhancing the selectivity andefficacy of anticancer block polymers for a wide array of cancer celllines.

The disclosed techniques take advantage of natural molecular traffickingmechanisms in cancer and pathogen derived diseases to enhance theefficacy of macromolecular therapeutics. In particular, the disclosedtechniques employ unique methods to modify antimicrobial and anticancerpolymers to target a broad spectrum of both pathogens and cancer celllines and enhance the transport of the respective polymers through thebacterial cell and/or the cancer cell membrane. In various embodiments,similar materials/mechanisms can be used to modify both antimicrobialand anticancer polymers to enhance the selective uptake of thesepolymers into the bacteria and/or cancer cell and thereafter cause therespective cells to undergo necrosis or autophagy. For example, withrespect to bacteria cells, in response to ingestion of the disclosedmodified antimicrobial polymers by bacterial cells, the bacterial cellsactivate reactive oxygen species (ROS) which cause cellular necrosis.With respect to anticancer cells, in response to ingestion of thedisclosed modified anticancer polymers by cancer cells, the anticancerpolymers activate autophagy, thereby eliminating the exposed cancercells.

In one or more embodiments, the uptake of polymer therapeutics in abroad spectrum of pathogens and cancer cells is enhanced via theintroduction of biotin. The added biotin can also enhance thetherapeutic activity of the polymers. In other embodiments, theselectivity of antimicrobial and anticancer polymers can besignificantly enhanced by the introduction of an anionic polymer incombination with the cationic therapeutic polymer to form a coacervate.In this regard, an anionic polymer (optionally with functional groups)can be combined with the cationic therapeutic polymer to generate anelectrostatic coacervate complex that is neutral and shields the toxicantimicrobial/anticancer cationic polymer when the complex circulatesthroughout the body, thereby reducing the toxicity of the cationicpolymer to mammalian cells. These coacervate complexes are well-definednanocomplexes that are highly modular with tunable particle size andneutral charge and remain stable under physiological conditions even inthe presence of serum proteins. In some implementations, the anionicpolymer and/or the cationic polymer can be functionalized with biotin tofurther increase the uptake of the coacervate complex by the pathogenand/or cancer cells. In one or more additional embodiments, thesecoacervate complexes can be used for diagnostic purposes. With theseembodiments, the cationic polymer can be calibrated to target a specificpathogen or cancer cell type, and the anionic polymer and/or thecationic polymer can be functionalized with a fluorescent dye thatilluminates in response to reaction of the coacervate with the specificpathogen or cancer cell type.

One or more embodiments are now described with reference to thedrawings, wherein like referenced numerals are used to refer to likeelements throughout. In the following description, for purposes ofexplanation, numerous specific details are set forth in order to providea more thorough understanding of the one or more embodiments. It isevident, however, in various cases, that the one or more embodiments canbe practiced without these specific details.

I—Biotinylated Therapeutic Polymers

In one or more embodiments, antimicrobial and anticancer therapeuticpolymers can be modified via functionalization with biotin (vitamin H)to facilitate the transport of these polymers through protein-basedchannels that traffic vitamins into bacteria as well as most cancer celllines that over express such channels. In this regard, bacteria andcancer cells readily accept biotin as a source of food/fuel. Byattaching biotin onto the therapeutic polymer, the bacteria/cancer cellsare essentially enticed by the biotin and ingest the entire polymer viatheir normal protein-based channels. Once inside the cell, theseantimicrobial/anticancer polymers initiate and/or facilitate killing ofthe cell.

As used herein, the term “biotinylated” refers to functionalization of amolecule, macromolecule, polymer, etc., with a biotin or biotin basedfunctional group. In various embodiments, the antimicrobial and/oranticancer therapeutic polymers that are biotinylated in accordance withthe disclosed techniques can include functional guanidinium polymers,referred to herein as polyguanidiniums. These polyguanidiniums can beformed using a controlled ROP of various cyclic carbonate monomersincorporating different antimicrobial and/or anticancer functionalities.These polyguanidiniums can comprise a hydrophobic polymer backboneconsisting of one or more covalently bonded polymer units, wherein atleast some (one or more) of the polymer units comprise a cationic(positively charged) guanidine-based functional group extendingtherefrom and covalently bonded to one or more atoms of the polymer unitvia a spacer group. In this regard, the polymer backbone can compriseone or more repeat monomer units that are respectively functionalizedwith a cationic, guanidine-based antimicrobial/anticancer moiety. Thesemonomers are referred to herein as guanidinium functionalized monomers.In various embodiments, the polyguanidiniums can be biotinylated via theattachment of biotin or a biotin based functional group (e.g., biotinol)to one or more ends or side chains of the polymer backbone. Apolyguanidinium comprising a biotin or biotin based functional group isreferred to herein as a biotinylated polyguanidinium (BG). In someembodiments, the biotin can be attached during ROP of the monomer usedto generate the biotinylated polyguanidinium. In other embodiments, thebiotin can be attached via post-polymerization modification of thebiotinylated therapeutic polymer.

In various embodiments, the subject biotinylated polyguanidiniums canfacilitate killing bacterial cells and/or cancer cells via membranetranslocation facilitated in part by the cationic guanidinium-moiety andfurther enhanced by the biotin functional group. For example, in someimplementation, when the disclosed biotinylated polyguanidinium are usedas an antimicrobial agent, the cationic guanidinium moieties can bindwith the anionic (negatively charged) phosphate groups on the bacterialcell membrane surface and a counterion exchange occurs between theguanidinium and the phosphate groups. As a result, the polymer becomesneutrally charged, allowing the polymer to translocate through the lipidbilayer of the bacterial membrane (e.g., as a non-polar species). Thepolymer is then released through the membrane leading to cytosolmaterial precipitation and subsequent cell necrosis. In this regard,release of the polymer into the bacteria cytosol cause precipitation ofthe biomacromolecules inside the cell of the bacteria, includingribonucleic acid (RNA), deoxyribonucleic acid (DNA), proteins, enzymes,etc. and the cell begins to kill itself by generating reactive oxygenspecies (ROS) which cause cellular necrosis. The disclosed biotinylatedpolyguanidinium can perform a same or similar translocation mechanismattributed in part to the cationic guanidinium moiety to facilitateentry of the polymer into cancer cells. In response to ingestion of thebiotinylated guanidinium polymers by cancer cells, the anticancerpolymers activate autophagy, thereby eliminating the exposed cancercells.

The biotin or biotin based functional group can further facilitate thetranslocation of the biotinylated polyguanidinium into the bacteria celland/or cancer cell by taking advantage of natural molecular traffickingmechanisms in cancer and pathogen derived diseases. In particular, thebiotin functional group opens up pore channels in both bacterial andcancer cells to facilitate increased uptake of the biotinylatedpolyguanidinium into the cell. In this regard, the biotin can in partserve as a molecular transporter. Based in the increased intracellularuptake of the biotinylated polyguanidiniums, the therapeutic activityand resistance prevention of the biotinylated polyguanidiniums aresignificantly enhanced.

In various embodiments, the antimicrobial and/or anticancer therapeuticpolymers that are biotinylated in accordance with the disclosedtechniques can include polyguanidiniums having chemical Formula 1 below,wherein R₁ comprises biotin or a biotin based functional group.

In accordance with Formal 1, the biotinylated polyguanidinium comprisesa number “n” of repeating monomer units, (referred to herein asguanidinium functionalized monomer units or the monomer units). Each (orin some embodiments one or more) of the monomer units can comprise apolycarbonate group and a cationic guanidinium moiety attached to thepolycarbonate a spacer group R₂. The spacer group R₂ can vary and can beadapted to facilitate a specific function or antimicrobial/antibacterialproperty of the polymer. For example, in some implementations the spacergroup R₂ can be selected/adapted to target a specific pathogen or cancercell type. In another example, the spacer group R₂ can comprise a groupthat causes the entire polymer to self-assemble into a micellestructure, wherein the cationic guanidinium portion of the polymerbecomes shielded within the micelle structure. One suitable functionalgroup that can facilitate this self-assembly can include a butyl group.Other suitable functional groups that can be employed for R₂ can includebut are not limited to: an alkyl group, an ethyl group, an isopropylgroup, a propyl group, a pentyl group, a cyclohexyl group, a phenylgroup, and a benzyl group.

In various exemplary embodiments, the disclosed antimicrobial/anticancerbiotinylated polyguanidiniums having Formula 1 can be or include abiotinylated polyguanidinium having Formula 2.

In accordance with Formula 2, the guanidinium moiety is attached to thepolymer backbone via an alkyl C₂ spacer group (e.g., R₂=an alkyl group)and the biotin functional group R₁ comprises biotinol. In one or moreembodiment, the chemical name for the polymer represented by Formula 2is Biotinol-[C2Gua]₁₇.

In other embodiments, the disclosed antimicrobial/anticancerbiotinylated polyguanidiniums can include block copolymers. For example,in one or more embodiments, having can be or include a biotinylatedpolyguanidinium copolymer having Formula 3, wherein R₁ comprises biotinor a biotin based functional group (e.g., biotinol) and wherein m=2 or1<m<20.

With reference to Formulas 1, 2 and 3 the number “n” and/or “m” ofrepeating and connected/bonded guanidinium functionalized monomer unitscan vary. For example, in some implementations, the number “n” ofrepeating monomer units can be one or more and one thousand or less.However, in various embodiments, the number “n” of repeating monomerunits can be less than 50 and more preferably less than 40 to reduce theparticle size of the polymer which facilitates better circulation andupdate in-vivo. In one implementation, the number “n” can be between 10and 40 units. In another implementation, the number “n” can be between10 and 40 units. With respect to Formula 3, in various embodiments, thenumber “m” can be less than the number “n”. For example, in someimplementations, the number “m” can be between 1.0 and 10.

In some implementations, the number “n” of repeating monomer units canbe tailored to balance the hydrophobicity of the polymer backbone groupand the spacer group R₂ relative to the hydrophilicity of theguanidinium moiety. In other implementation in which R₂ comprises abutyl group, the number “n” of repeating monomer units can be tailoredto facilitate formation of the subject biotinylated polyguanidinium intoprotected micelle nanostructures in aqueous solution to facilitate theself-assembly of the subject polymers into the protected micellenanostructures, wherein the guanidinium moieties are exposed on theoutside of the micelle on the out and the hydrophobic residuals areinternalized within a micelle. In this regard, in some embodiments, oneor more polymers having Formula 1 can be configured to self-assembleinto the protected micelle nanostructures when R₂ comprises a butylgroup and “n” is between 10 and 50, and more preferably between 20 and40.

FIG. 1 presents an example synthesis route 100 for generating abiotinylated polyguanidinium having chemical Formula 2 in accordancewith various embodiments described herein. Synthesis route 100facilitates the attachment of biotin to therapeutic polymers via themodification of biotin into an alcohol form, which is then repurposed asa polymerization initiator. In this regard, synthesis route 100facilitates the attachment of a biotin or biotin based functional groupto the therapeutic monomer in association with the ROP to form thepolymer.

In the embodiment shown, synthesis route 100 is divided into a two-partprocess, wherein biotinol 108 is generated in accordance with synthesisroute 100A, and thereafter in accordance with synthesis route 100B, thebiotinol 108 is attached to the polyguanidinium in association with theROP of the cyclic guanidinium monomer 110. With reference to synthesisroute 100A, a biotin monomer 102 can be reacted with oxalyl chloride 104at room temperature (r.t.) to modify the biotin monomer 102 intocompound 106. Compound 106 can further be reduced at r.t. to formbiotinol 108, which is a modified alcohol form of the biotin monomercomprising a hydroxyl group side chain. In accordance with synthesisroute 100B, this biotinol 108 can be used as an initiator of a ROP ofthe cyclic guanidinium monomer 110 in association withreagents/catalysts including 1,8-Diazabicyclo[5.4.0]undec-7-ene/thiourea(DBU/TU) and methylene chloride (CH₂CL₂) to form protected biotinylatedpolyguanidinium 114. The protected biotinylated polyguanidinium 114 canthen be deprotected using reagents/catalysts trifluoroacetic acid (TFA)and dichloromethane (DCM) in r.t. overnight to form the biotinylatedpolyguanidinium 116 (BG) having Formula 2.

Although synthesis route 100B is demonstrated using a single, cyclicguanidinium monomer 110 having an alkyl spacer group, synthesis route100B can be extended to other biotinylated polyguanidiniums. Forexample, synthesis route 100B can be used to attach biotin to variousbiotinylated polyguanidinium monomer and copolymer variations (e.g.,with different spacer groups for R₂, biotinylated polyguanidinium havingFormula 3, and the like) using biotinol 108 as the initiator for the ROPof the cyclic guanidinium monomer.

Furthermore, the disclosed techniques for enhancing update oftherapeutic polymers via the attachment of a biotin or biotin basedfunctional group thereto can be applied to anticancer and antimicrobialpeptides. In this regard, the disclosed biotinylated therapeuticpolymers are not limited to polyguanidinium polymers. For example, insome embodiments an anticancer and antimicrobial quaternary ammoniumfunctional block copolymer can be biotinylated (e.g., in accordance withsynthesis route 100 or synthesis route 200 described below) to furtherenhance the update of these polymers into tumor cells.

FIG. 2 presents another example synthesis route 200 for generating abiotinylated therapeutic polymer in accordance with various embodimentsdescribed herein. Synthesis route 200 facilitates the attachment ofbiotin to a therapeutic polymer via post-polymerization modification. Inthis regard, in some embodiments, functionality can be introduced to thedisclosed therapeutic macromolecules via post polymerizationmodification. The functionality can include a biotin based functionalgroup, a guanidinium based functional group, a sugar based functionalgroup, and the like. Post polymerization modification provides anefficient and straightforward manufacturing process that can be used tosynthesize functionalized therapeutics macromolecules with significantdiversity. For example, synthesis route 200 can be used to form atherapeutic polymer having chemical formula 4 below, wherein R₁ cancomprise a variety of functional groups that can be used as theinitiator of the ROP of the polymer, and R₂ comprises biotin or a biotinbased functional group. For example, in some embodiment, R₁ can comprisea benzyl alcohol, a disaccharide (a sugar), a dansyl amid (a reactivefluorescent dye), or another suitable hydroxyl ROP initiator.

In accordance with synthesis route 200, the therapeutic polymer 202comprises a perfluoro amine functional group as opposed to guanidinium.In various embodiment, the perfluoro amine can be cationic and providesame or similar cell translocation functionalities (e.g., via ionexchange) as the guanidinium moiety. In this regard, in some embodimentsof synthesis route 200, a guanidinium moiety can be used instead of theperfluoro amine. In order to attach the biotin functional group R₂ tothe therapeutic polymer 202, a thiol such as tetramethylsilane (TMSS)can be installed onto the biotin R₂ to generate the modified biotin 204.The modified biotin 204 can then be used to perform a nucleophilicaromatic substitution on the cationic moiety (e.g., the perfluoro amineor in some cases a guanidinium moiety or the like) to generate thebiotinylated therapeutic polymer 206 having chemical formula 4.

In accordance with Formula 1, Formula 2, Formula 3 and Formula 4, thepolymer backbone comprises polycarbonate. However, in one or moreadditional embodiments, other hydrophobic polymers can be employed asthe polymer backbone. For example, in other embodiments, the polymerbackbone can comprise polylysine, polyionene, polyethylenimine and thelike. In some implementations, no restriction is placed on the polymerskeletal structure of the skeletal backbone. Exemplary non-limitingpolymer skeletal structures can include linear polymers, branchedpolymers, star polymers, mykto-arm star polymers, latter polymers,cyclic polymers, and graft polymers. The forgoing polymer types cancomprise a homopolymer, a random copolymer, or a block copolymer chain.In various exemplary embodiments, the biotinylated polyguanidinium is alinear polymer comprising a plurality of covalently bonded guanidiniumfunctionalized monomer units. Herein, a linear polymer has one branchhaving two peripheral ends (i.e., dangling ends, as the two ends of asegment of a rope). The one branch can comprise one or more polymerchain segments covalently linked together at respective polymer chainends by way of any suitable linking group, which can include a singlebond. Each polymer chain segment of a linear polymer can comprise ahomopolymer, random copolymer, or block copolymer chain comprising oneor more repeat units. At least one of the polymer chain segmentscomprises one or more repeat units of a monomer comprising a cationicfunctional group, such as guanidinium functionalized monomer, perfluoroamine monomer, or the like.

The various biotinylated therapeutic polymers having chemical formulas1-4 discussed above have demonstrated strong, broad-spectrum,antimicrobial selectivity and efficacy toward a variety of gram-negativeand gram-positive bacteria types, including P. aeruginosa. Theantimicrobial efficacy and/or specificity (or the degree of selectivitytoward microbial cells as opposed to mammalian cells) of thebiotinylated form of these polymers relative to the same polymerswithout biotin was further increased. The biotinylated therapeuticpolymers having chemical formulas 1-4 have also demonstrated strong,broad-spectrum selectivity and efficacy against many different cancercell lines. Similarly, the antimicrobial efficacy and the degree ofselectivity toward cancer cells as opposed to mammalian cells of thebiotinylated form of these polymers relative to the same polymerswithout biotin was further increased.

For example, FIG. 3 presents transmission electron microscopy (TEM)micrograph images of a cancer cell before and after treatment with abiotinylated polyguanidinium based anticancer agent in accordance withvarious embodiments described herein. In accordance with this example,the anticancer agent used comprised a block copolymer formed withguanidinium monomers (e.g., having chemical Formula 3 or the like). Whenused as an anticancer agent, these polymers self-assemble into micelles.Upon interaction with cancer cells, these polymers translocate thoughthe cancer cell membrane as facilitated by the micelle structure, theguanidinium moiety and the biotin group, and accumulate within thecancer cell. Once inside, the accumulated polymers kill the cancer cellquite dramatically. For example, as shown in FIG. 3, the left TEM image(image 301) depicts a cancer cell prior to treatment with thepolyguanidinium block copolymer, and the TEM image on the right (image302), depicts the same cancer cell post treatment. As shown in image302, the polymers translocate into the cancer cell and cause autophagyvia vacuolization. For example, the polymer causes the cancer cell toform holes or vacuoles 303 as a result of the cell essentiallydestructing itself from the inside out.

FIGS. 4A and 4B present charts illustrating the in vitro efficacy of abiotinylated polymer and a non-biotinylated polymer against cancer cellline BT-474 (a human ductal carcinoma cell line) in accordance withvarious embodiments described herein. The biotinylated polymer testedcomprised a biotinylated polyguanidinium having chemical Formula 2 (BG).The non-biotinylated polymer tested for comparison comprised apolyguanidinium referred to herein in as Polymer B or 4m-[C2Gua]₁₇. Thechemical structure for Polymer B is shown in the upper righthand cornerof FIG. 4A with reference to arrow 400. In accordance with FIGS. 4A and4B, the anticancer efficacy of the respective polymers was tested as afunction of cell viability using a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayat different concentrations over a 2 day (48 hour) period. FIG. 4Apresents a Chart 401 demonstrating the results after 1 day (24 hours)and FIG. 4B presents a Chart 402 demonstrating the results after 2 days(48 hours). As shown in Charts 4A and 4B, the BG (biotinylatedpolyguanidinium) has significantly stronger anticancer activity relativeto the non-biotinylated polyguanidinium as evidenced by the lower amountof anticancer agent required to inhibit survival (or kill) of 50% of thecancer cell population, referred to as the IC50 value. In this regard,after 1 day, the IC50 value for BG 31 μg/ml is half that of m4 which is250 μg/ml.

FIG. 5 presents a Chart 500 illustrating the cytotoxicity of abiotinylated polymer against healthy non-cancerous mammalian cell lineHEK293 in accordance with various embodiments described herein. Thebiotinylated polymer tested comprised a biotinylated polyguanidiniumhaving chemical Formula 2 (BG). In accordance with Chart 500, thecytotoxicity of BG against HEK293 was tested as a function of cellviability using an MTT assay. As shown in Chart 500, almost 100% of theHEK293 cells survived at concentrations of BG up to about 31 μg/ml. BGfurther showed a high IC50 value at about 125 μg/ml, rending theselectivity of BG toward HEK293 125/31 or 4 μg/ml. This demonstratesthat the disclosed biotinated polyguanidiniums not only demonstrate highefficacy as an anticancer agent, but also demonstrate strong selectivitytoward diseased cells over healthy mammalian cells.

FIG. 6 presents a high-level flow diagram of an example method 600 forenhancing the selectivity and efficacy of therapeutic polymers against abroad spectrum of pathogens and cancer cell lines via the introductionof biotin in accordance with various embodiments described herein.Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

At 602, a therapeutic polymer can be formed based on polymerization of aplurality of therapeutic monomers (e.g., cyclic carbonate guanidiniumfunctionalized monomers), wherein the therapeutic polymer provides atherapeutic functionality (e.g., an antimicrobial and/or anticancerfunctionality). At 604, biotin can be attached to the therapeuticpolymer, resulting in a biotin-functionalized therapeutic polymer (e.g.,BG), wherein the biotin-functionalized therapeutic polymer providesgreater therapeutic efficacy relative to the therapeutic polymer. Forexample, in some embodiments, the biotin can be attached via synthesisroute 100. In other embodiments, the biotin can be attached viasynthesis route 200.

II—Therapeutic Coacervates

In various additional embodiments, to improve selectivity,bioavailability and reduce serum complexation, an anionic carrier and beused to deliver the antimicrobial and/or anticancer polymers in the forma coacervate. Coacervation is a phenomenon in which cationic and anionicwater-soluble polymers interact in fluid (e.g., water, serum, etc.) toform a liquid, polymer-rich phase complex held together by electrostaticforces. The term “conciliate” is used herein to refer to the complexformed between two polymers as a result of coacervation. In accordancewith the disclosed techniques, a coacervate can be formed between acationic antimicrobial polymer and an anionic carrier polymer. Thecoacervate complex shields the cationic therapeutic polymer in a looseparticle structure based on the dynamic electrostatic interactionbetween the cationic polymer and the anionic polymer, thereby reducingtoxicity of the cationic therapeutic polymer mammalian cells.

In this regard, the coacervate complexes disclosed herein are relativelyloose particle structures held together by electrostatic forces. Thecoacervate complexes are further neutral in charge as a result of chargecancelation between the anionic and cationic polymer. As a result, theanionic polymer and the cationic therapeutic polymer remain in a dynamicequilibrium when suspended in solution. However, when the coacervatecomplex interacts with the negatively charged surface the cellularmembrane of a bacteria cell or cancer cell, the coacervate complex opensup to expose the cationic therapeutic polymer because the cationictherapeutic polymer is more attracted to the bacteria or cancer cellmembrane relative to the anionic carrier polymer. As a result, thecationic therapeutic polymer is released from the coacervate complex andan ion exchange occurs between the cationic therapeutic polymer and theanionic surface of the bacteria or cancer cell. This ion exchangeneutralizes the cationic polymer and facilitates translocation of thecationic polymer through the bacteria or cancer cell membrane. Onceinternalized, the cationic polymer induces necrosis by the bacterialcell or autophagy by the cancer cell, respectively.

FIG. 7 presents an example synthesis route 700 for forming a therapeuticcoacervate in accordance with various embodiments described herein. Inaccordance with synthesis route 700, an anionic polymer 702 can becombined with a therapeutic cationic polymer 704 in solution (e.g.,water, DI water, serum, bodily fluids, etc.) to form coacervate complex706. In the embodiment shown, the anionic polymer 702 comprises asulfonate functional polycarbonate having chemical Formula 5 below andreferred to herein as Polymer A. The therapeutic cationic polymer 704comprises a functional guanidinium cationic copolymer having chemicalFormula 6 below and referred to herein as Polymer E.

The coacervate complex 706 formed between anionic Polymer A and cationicPolymer E is referred to herein as Complex E. As shown in FIG. 7, theanionic polymer 702 can comprise an anionic polymer backbone 710 and arelative neutral polyethylene glycol (PEG) tail 708 extending therefrom.In the embodiment shown, the PEG is more specifically methoxypolyethylene glycol (MPEG). In various embodiments, the PEG tail 708 cancomprise MPEG or PEG. Based in part on this chemical structure of theanionic polymer 702, the combined anionic polymer and cationic polymerself-assemble into coacervate complex 706 having a relativelycircular/spherical particle structure. The structure of the coacervatecomplex 706 shields the cationic portion 712 of the cationic polymerwithin an internal region of the particle, surrounded and protected bythe neutral, PEG tail 708 portion of the anionic polymer 702.

For example, FIG. 8 provides a transmission electron microscopy (TEM)image of Complex E in accordance with various embodiments describedherein. As shown in FIG. 8, the coacervate complexes 706 have arelatively circular or spherical structure around 100 nm in diameter.

With reference again to FIG. 7, the PEG tail 708 facilitates shieldingthe cationic portion 712 of the therapeutic cationic polymer 704 withinthe formed coacervate complex. The PEG tail 708 also facilitatescirculation of the coacervate complex 706 in solution (e.g., within thebody) and minimizing the toxicity of the coacervate complex 706 towardmammalian cells. In this regard, PEG is water soluble and the human bodydoesn't really recognize PEG. When the anionic polymer 702 and thetherapeutic cationic polymer 704 assemble into the coacervatenanoparticles, the PEG presents itself on the outside. As a result,macrophages and biomolecules that normally sequester particles recognizethe coacervate complex 706 only as water. Thus, immune cells don'tsequester the coacervate complex or try kill it.

The number “m” of PEG units and the number “n” of polymer units thatmake up the polymer backbone of Polymer A can vary. In the embodimentshown, the PEG tail 708 is identified as MPEG 10K, which denotes amixture of PEG molecules (about 195-265 PEG molecules) having an averageMW of 10,000 g/mol. In this regard, the value of m can be between about195 and 265. However, in other embodiments, m can be between 40 and 500.In various embodiment, the value of “n” for Polymer A can be between 10and 100, more preferably between 20 and 80, and even more preferablybetween 30 and 50. In various embodiments, the value of “n” for PolymerA is 40. The value of “n” and “m” with respect to Polymer E can alsovary. For example, in some embodiments, the value of n for Polymer E canbe less than 50 and more preferably less than 41, and in one embodiment,the value of n in for Polymer E is 11. The value of m for Polymer E canbe less than 10 and more preferably less than 5. In various embodiments,the value of m for Polymer E can be 2.

Complex E provides one example coacervate complex that provides bothantimicrobial and anticancer functionality with higher efficacy and/orselectivity relative to the cationic Polymer E alone. Various additionalantimicrobial and anticancer coacervates can be formed in accordancewith synthesis route 700 using other anionic polymers in combinationwith Polymer E, as well as other therapeutic cationic polymers. Forexample, some additional anionic polymers that can be used instead ofPolymer A to form a therapeutic coacervate complex in combination withPolymer E or another therapeutic cationic polymer can includeacid-functionalized polycarbonates such a carboxylic acid-functionalizedpolymer, a phosphoric acid-functionalized polymer, or the like. In thisregard, one example acid-functionalized polymer that can be used as theanionic polymer instead of Polymer A can include a diblock carboxylicacid functionalized copolymer having chemical Formula 7 below andreferred to herein as Polymer J:

In accordance with Formula 7, the number “m” of PEG units and the number“n” of polymer units that make up the polymer backbone of Polymer J canvary. In this regard, the value of m can be between about 195 and 265.However, in other embodiments, m can be between 40 and 500. In oneexemplary embodiment, m can be 113. In various embodiment, the value of“n” for Polymer J can be between 10 and 100, more preferably between 20and 80, and even more preferably between 30 and 50. In variousembodiments, the value of “n” for Polymer J is 40.

Furthermore, in some embodiments, biotin ligands can be installed on oneor more chain ends of the anionic polymer 702 and/or the therapeuticcationic polymer 704 to enhance the targeting selectivity of theresulting coacervate complex. For example, in some embodiments, PolymerE can be replaced with the polymer having chemical Formula 2 (alsoreferred to herein as Polymer BG). In other embodiments, the anionic(e.g., Polymer A, Polymer J, and the like) can be biotinylated via theattachment of a biotin group to one or more ends of the polymer backboneand/or a side chain extending from the polymer backbone. In accordancewith these embodiments, the biotinylated form of Polymer A is referredto herein as Polymer A′ and has chemical Formula 8 below, and thebiotinylated form of Polymer J is referred to herein as Polymer J′ andhas chemical Formula 9 below.

As shown with reference to Formulas 5 and 8, Polymer A′ can comprise asame or similar structure as Polymer A with the addition of a biotinolfunctional group to an end of the polymer backbone. In this regard, insome embodiments, “m” and “n” can be the same values in Formula 8 asthose used for Formula 5. In some embodiments however, the values for“m” and “n” for Polymer A′ can be different than those used for PolymerA. Similarly, Polymer J′ can comprise a same or similar structure asPolymer J with the addition of a biotinol functional group to an end ofthe polymer backbone. In this regard, in some embodiments, “m” and “n”can be the same values in Formula 9 as those used for Formula 7. In someembodiments however, the values for “m” and “n” for Polymer J′ can bedifferent than those used for Polymer J.

Other therapeutic (e.g., anticancer and/or antimicrobial) cationicpolymers that can be used instead of Polymer E can include but are notlimited to Polymer B, Polymer C, Polymer F, Polymer D, Polymer G andPolymer H, respectively having chemical Formulas 10, 11, 12, 13, 14 and15 as follows:

The values of “n” and “m” with respect to the cationic polymers havingFormulas 10-15 can vary. In one or more embodiments, with respect toFormula 10 (Polymer B), n can be less than 60 and more preferably lessthan 45. In one embodiment, the value of n in Formula 10 can be 16.Similarly, with respect to Formula 11 (Polymer C), n can be less than 60and more preferably less than 45, and in one embodiment, the value of nin Formula 11 can be 16. With respect to Formulas 12 and 15 (Polymer Fand Polymer H, respectively) can be less than 60 and more preferablyless than 45, and in one embodiment, the value of n in Formula 12 can 19and the value of n in Formula 15 can be 20. With respect to Formula 13(Polymer D) and Formula 14 (Polymer G), n can be less than 60 and morepreferably less than 45, and in one embodiment, the value of n inFormulas 13 and 14 can be 16. The value of m in Formulas 13 and 14 canbe less than 10 and more preferably less than 5. In various embodiments,the value of m in Formulas 13 and 14 can be 2.

In this regard, in addition to Complex E, in various embodiments,antimicrobial and/or anticancer coacervate complexes can be formed usinga combination of an anionic polymer such as Polymer A, Polymer A′,Polymer J, Polymer J′ or the like, with a cationic polymer, wherein thecationic polymer can include but is not limited to, one of: Polymer B,Polymer C, Polymer D, Polymer E, Polymer F, Polymer G, Polymer H andPolymer BG (wherein Polymer BG has chemical Formula 2 supra). Theresulting coacervate complexes are respectively referred to herein asidentified in Table 1.

TABLE 1 Anionic Polymer Cationic Polymer Coacervate Polymer A Polymer BComplex B Polymer A′ Polymer B Complex B′ Polymer A Polymer C Complex CPolymer A′ Polymer C Complex C′ Polymer A Polymer D Complex D Polymer A′Polymer D Complex D′ Polymer A Polymer E Complex E Polymer A′ Polymer EComplex E′ Polymer A Polymer F Complex F Polymer A′ Polymer F Complex F′Polymer A Polymer G Complex G Polymer A′ Polymer G Complex G′ Polymer APolymer H Complex H Polymer A′ Polymer H Complex H′ Polymer A Polymer BGComplex BG Polymer A′ Polymer BG Complex BG′ Polymer J Polymer B ComplexBJ Polymer J′ Polymer B Complex BJ′ Polymer J Polymer C Complex CJPolymer J′ Polymer C Complex CJ′ Polymer J Polymer D Complex DJ PolymerJ′ Polymer D Complex DJ′ Polymer J Polymer E Complex EJ Polymer J′Polymer E Complex EJ′ Polymer J Polymer F Complex FJ Polymer J′ PolymerF Complex FJ′ Polymer J Polymer G Complex GJ Polymer J′ Polymer GComplex GJ′ Polymer J Polymer H Complex HJ Polymer J′ Polymer H ComplexHJ′ Polymer J Polymer BG Complex BGJ Polymer J′ Polymer BG Complex BGJ′

FIG. 9A presents an example synthesis route 900 for forming Polymer A inaccordance with various embodiments described herein. In the embodimentshown, a tritol (Tr) protected thiol monomer 902 can be reacted withreactants 904 including MPEG 10K, DBU/TU and CH₂CL₂ to form the blockcopolymer 906. In this regard, using MPEG as an initiator, a ROP of thecyclic carbonate of protected thiol monomer 902 can be performed togenerate the block copolymer 906 including the protected thiol with theTr group. Tr represents three phenyl groups. These phenyl groups can bedeprotected resulting in an unprotected sulfur-hydrogen (SH) thiol. Inthis regard, the block copolymer 906 can further be reacted withreactants 910, including triisopropylsilane (TIPS),meta-chloroperoxybenzoic acid (MCPBA) and TFA at r.t., to deprotect theTr group and cause an oxidation of the thiol to generate the sulfonatepolymer 912, referred to herein as Polymer A.

FIG. 9B presents an example synthesis route 901 for forming Polymer A′in accordance with various embodiments described herein. Synthesis route901 is similar to synthesis route 900 with the addition of a biotinfunctional group to the PEG 10K used for the ROP of the protected thiolmonomer 902. Repetitive description of like elements employed inrespective embodiments is omitted for sake of brevity. In accordancewith synthesis route 901, the Tr, protected thiol monomer 902 can bereacted with reactants 914 including biotinylated PEG, DBU/TU and CH₂CL₂to form the block copolymer 916. In this regard, using biotinylated PEGas an initiator, a ROP of the cyclic carbonate of protected thiolmonomer 902 can be performed to generate block copolymer 916 includingthe Tr group and the biotinol functional group bound to an end of theblock copolymer 916. The block copolymer 916 can further be reacted withreactants 910, including TIPS, MCPBA and TFA at r.t., to deprotect theTr group and cause an oxidation of the thiol to generate thebiotinylated sulfonate polymer 918, referred to herein as Polymer A′.

FIG. 9C presents an example synthesis route 903 for forming Polymer J inaccordance with various embodiments described herein. Synthesis route903 is described as a two-part reaction including route 903A followed byroute 903B. With reference to route 903A, in the embodiment shown, aprotected cyclic carboxylic acid monomer 920 can be reacted withreactants 922 including PEG, DBU/TU and CH₂CL₂ to form block copolymer924 with a protected carboxylic acid group. In this regard, using thePEG as an initiator, a ROP of the protected cyclic carboxylic acidmonomer 920 can be performed to generate the block copolymer 924including a protected carboxylic acid group. In accordance with route903B, the block copolymer 924 can further be reacted with reactants 926,including CF₃COOH (trifluoroacetic acid) and CH₂Cl₂ at r.t., todeprotect the carboxylic acid group to generate the diblock acidfunctionalized polycarbonate referred to herein as Polymer J.

FIG. 9D presents an example synthesis route 905 for forming Polymer J′in accordance with various embodiments described herein. Synthesis route905 is described as a two-part reaction including route 905A followed byroute 905B. Synthesis route 905 is similar to synthesis route 903 withthe addition of a biotin functional group to the PEG used for the ROP ofthe protected carboxylic acid monomer 920. Repetitive description oflike elements employed in respective embodiments is omitted for sake ofbrevity. In accordance with synthesis route 905A, the protectedcarboxylic acid monomer 920 can be reacted with reactants 930 includingbiotinylated PEG, DBU/TU and CH₂CL₂ at r.t. to form the block copolymer932. In this regard, using the biotinylated PEG as an initiator, a ROPof the protected cyclic carboxylic acid monomer 920 can be performed togenerate the block copolymer 922 including a protected carboxylic acidgroup and a biotinol group. In accordance with route 905B, the blockcopolymer 923 can further be reacted with reactants 926, includingCF₃COOH (trifluoroacetic acid) and CH₂Cl₂ at r.t., to deprotect thecarboxylic acid group to generate the biotinylated diblock acidfunctionalized polycarbonate referred to herein as Polymer J′.

FIG. 10 presents example synthesis routes for forming functionalguanidinium cationic homopolymers Polymer B, Polymer C and Polymer F inaccordance with various embodiments described herein. In the embodimentshown, the respective synthesis routes are identified as route 1000A,route 1000B and route 1000C. Route 1000A can be used to form Polymer B,route 1000B can be used to form polymer C and route 1000C can be used toform Polymer F. Each of these routes involve a ROP of guanidiniummonomer 1002 with a different chemical initiator (and catalysts DBU, TUand solvent CH₂CL₂) to generate the corresponding protected homopolymers1004, 1006 and 1008. For example, the initiator used with route 1000Acomprises benzyl alcohol, the initiator used with route 1000B comprisesa disaccharide (e.g., mannose), and the initiator used with route 1000Ccomprises a dansyl amid (a reactive fluorescent dye). The protectedhomopolymers 1004, 1006 and 1008 respectively, can subsequently bedeprotected using TFA and DCM to generate the corresponding functionalguanidinium cationic homopolymers including Polymer B, Polymer C andPolymer F, respectively.

FIG. 11 presents example synthesis routes for forming functionalguanidinium cationic copolymers, Polymer D, Polymer E and Polymer G, inaccordance with various embodiments described herein. Repetitivedescription of like elements employed in respective embodiments isomitted for sake of brevity.

In the embodiment shown, the respective synthesis routes are identifiedas route 1100A, route 1100B and route 1100C. Route 1100A can be used toform Polymer D 1104, route 1100B can be used to form polymer E 1106, androute 1000C can be used to form Polymer G 1108. Routes 1100A, 1100B, and1100C are similar to routes 1000A, 1000B and 1000C, respectively, withthe addition of a urea monomer 1102 with the guanidinium monomer 1002.The urea monomer 1102 comprises a urea group (NH-carbonyl-NH). The ureamonomer 1102 enhances the kinetic stability of the resulting functionalguanidinium cationic copolymers (e.g., Polymer D, Polymer E, and PolymerG, respectively) and the subsequent coacervates that are formed when thecationic polymers are combined with an anionic polymer. The amount ofurea monomer used can be less than the amount of guanidinium monomerused to form the respective polymers. For example, in the embodimentsshown, the respective polymers can include 11 or 16 guanidinium monomerunits and only 2 urea monomer units.

FIG. 12 provides a Table 1200 identifying physical characteristics ofexample therapeutic coacervate complexes in accordance with variousembodiments described herein. In particular, Table 1200 identifies theparticle size, the polydispersity index (PDI) and the zeta potential ofvarious antimicrobials, including Complexes B, C, D, E and E′.Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

The coacervates formed between one or more of the disclosed cationictherapeutic polymers and anionic carriers are well-defined complexeshaving a particle size less than 200 nm, 150 nm or 100 nm. In thisregard, the coacervate complexes disclosed herein can be characterizedas a nanocomplex or nanoplex. For example, as shown in Table 1200,Complex B exhibits a particle size at or near 132.0±1 nm, Complex Cexhibits a particle size at or near 117.0±1 nm, Complex D exhibits aparticle size at or near 138.0±1 nm, Complex E exhibits a particle sizeat or near 82.0±1 nm, and Complex E′ exhibits a particle size at or near71.0±1 nm. The small particle size of the subject coacervate complexesfacilitates circulation of the coacervates and inhibits sequestering bymammalian cells in vivo. The small particle size further facilitatesuptake of the complexes by cancer cells via the enhanced permeationretention effect (EPR). For example, as a tumor multiplies, the vasculararound the tumor opens up, leaving holes that the small coacervateshaving a size less than 200 nm can fall into. Accordingly, thenanoparticle size of the subject coacervate complexes facilitates thetransport of the polymer complex into the cancer/tumor cell forintracellular killing.

The respective complexes further exhibit low PDI. For example, Complex Eand E′ demonstrate a very narrow size distribution with a PDI of about0.08±0.02 nm. Complexes B, C and D also exhibit low PDIs. Thepolydispersity index (PDI) (or more recently referred to as dispersityindex), provides a measure of the distribution of molecular mass in agiven polymer sample of the particles sizes. The lower the PDI, the moreuniform the distribution of the particle size. A low PDI facilitates theantimicrobial and/or anticancer efficacy and/or selectivity of thesubject coacervate complexes. For example, high PDI reflects a mixtureof particles with vast size variation (some very large, some verysmall), which weakens the rate of update or permeation of the complexesintracellularly.

The zeta potential is a measure of electric charge associated with amolecule or macromolecule. The closer the zeta potential to zero, themore neutrally charged the complex, thereby promoting bettercirculation, and inhibiting in vivo sequestering by macrophages andbiomolecules. Accordingly, a zeta potential close to zero is preferred.As shown in Table 1200, the subject Complexes respectively demonstraterelatively neutral zeta potentials, with Complex E and Complex E′demonstrating the lowest.

Complex E and Complex E′ are highlighted in Table 1200 because theseparticular Complexes demonstrate higher overall physical characteristicsrelative the other coacervate Complexes. For example, complex E exhibitsa particle size at or near 82.0±1 nm, with a very narrow sizedistribution or PDI of about 0.08±0.02, and a Zeta potential that issubstantially zero, a −0.01±0.3 mV. Importantly, lyophilized Complex Egave similar size (97±4 nm) to its aqueous dispersion form (82±1 nm),and was able to re-disperse in water easily without usingcryoprotectants. Complex E′ exhibits similar PDI and Zeta potentialcharacteristics as Complex E, with an even smaller particle size, at ornear 72.0±1. Accordingly, Complex E and Complex E′ are highly promisingtargets for antimicrobial and/or anticancer agents. Several exampleexperiments described infra evaluating the selectivity and efficacy ofthe subject coacervate Complexes use Complex E and/or Complex E′ as aprimary example based in part on the superior physical propertiesexhibited by these complexes.

In addition to the excellent physical characteristics of the subjectcoacervate complexes described with reference to Table 1200, thecoacervate complexes demonstrate strong kinetic stability underphysiological conditions even in the presence of serum proteins.

FIGS. 13A and 13B present graphs illustrating the kinetic stability ofexample coacervates in accordance with various embodiments describedherein. The coacervates measured in graphs shown in FIGS. 13A and 13Binclude coacervates formed with anionic Polymer A, and respectivecationic polymers B, C, D and E when mixed in a solution comprisingdeionized water (DI) with 10% fetal bovine serum. The coacervates formedare respectively include Complex B, Complex C, Complex D, and complex E.Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

FIG. 13A presents a Graph 1301 demonstrating the change in coacervatesize over time after the respective complexes are initially formed inthe solution. As shown in Graph 1301, the size of the respectivecoacervates remained below 210 nm over a 24-hour period. This indicatesthat the respective complexes did not interact with and/or were notdisrupted by biomolecules present in the fetal bovine serum. Thus, therespective coacervates demonstrate strong kinetic stability within serumwithout sequestering or interaction with serum proteins.

FIG. 13B presents a Graph 1302 demonstrating the change in scatteredlight intensity over time measured after addition of Triton-X to the DIwater in which the respective coacervates are formed. The Triton-X isused to intentionally stress or destabilize the complexes to test theirstability as a function of resistance to the Triton-X over time. In thisregard, addition of the Triton-X to the DI water after formation of thecomplexes therein emulates the in vivo environment the complexes wouldencounter when injected into a mammalian model (e.g., from a solutioninto the body, a phenomenon referred to as infinite dilution). In Graph1302, the relative intensity (%) is represented as the percentage ofscattered light intensity at a variable time point relative to scatteredlight intensity at time h=0. As shown in Graph 1302, all of the measuredcoacervates (e.g., Complex B, Complex C, Complex D and Complex E),remained intact or substantially intact (e.g., did not disassemble) overa 24-hour period. This simulated experiment indicates that the disclosedcoacervate complexes demonstrate strong kinetic stability in an in vitroenvironment.

FIG. 14 presents a Graph 1400 illustrating the in vitro release profileof cationic polymers from some example coacervates in accordance withvarious embodiments described herein. Graph 1400 depicts the results ofan experiment in which coacervates including Complex F and Complex Gwere mixed in a phosphate buffered-saline (PBS) solution at 7.4 pH and5.8 pH under simulated shaking conditions at 37° Celsius (C). Thisexperiment tests the release of polymer F and G from the respectivecoacervates Complexes F and G as a function of time. As shown in Graph1400, with respect to lines 1401, 1402, and 1403, release of Polymer Fat a pH of 7.4 and Polymer G at a pH of 7.4 and 5.8 was slow over a120-hour period. As indicated by line 1404, release of Polymer F at 5.8pH was relatively higher over the 120-hour period, with a cumulativerelease less than 40%. Although the overall release of the cationicpolymers was slow, they would dissociate from the complexes to bindanionic phosphate groups on bacterial membrane surface upon contact withbacteria and translocate across the membrane.

The various coacervate complexes disclosed herein, such as thoseidentified in Table 1 and similar variations, have demonstrated strongselectivity and antimicrobial efficacy and specificity toward a varietyof bacteria types including P. aeruginosa. The antimicrobial efficacyand/or specificity (or the degree of selectivity toward bacteria cellsas opposed to mammalian cells) of the coacervate form of the variouscationic therapeutic polyguanidiniums (e.g., Polymer B, Polymer C,Polymer D, Polymer, E, Polymer F, Polymer G, Polymer H and Polymer BG)when combined with an anionic Polymer (e.g., Polymer A, Polymer A′,Polymer J, Polymer J′ and the like) relative to the same cationicpolyguanidiniums polymers alone was further increased. The coacervatecomplexes identified in Table 1 and variations thereof have alsodemonstrated strong specificity and efficacy as an anticancer agent formany different cancer cell lines. Similarly, the degree of selectivityof the coacervates toward cancer cells as opposed to mammalian cells andthe degree of efficacy of the coacervates against cancer cells relativeto their corresponding cationic polymers alone was further increased.FIGS. 15A-23 provide evidence-based data demonstrating these therapeuticproperties and the disclosed coacervate complexes.

FIG. 15A presents a Graph 1500 illustrating the antimicrobialselectivity and efficacy of example therapeutic polymers and coacervatecomplexes against P. aeruginosa in accordance with various embodimentsdescribed herein. In accordance with Graph 1500, individual polymersincluding Polymer A, Polymer B, Polymer C, Polymer D along withcoacervates including Complex B, Complex C, Complex D and Complex E weretested for efficacy against P. aeruginosa. The tested efficacy is basedon their respective minimal inhibitory concentrations (MICs) toward P.aeruginosa. Their selectivity/toxicity toward rat red blood cells (RBCs)was also tested, measured in hemolysis concentration (HC) 50 or (HC50),which corresponds to the amount of antibacterial agent required to kill50% of the RBCs. The commercial standard antibiotic Polymyxin B sulfate(PolyB) and Complex X were also tested for selectivity andefficacy/toxicity for comparison. Complex X is used herein to refer to acoacervate complex formed with Polymer A in combination with PolyB (asopposed to Polymer A and the disclosed therapeutic polyguanidinium basedPolymers). FIG. 15B provides a corresponding Table 1501 for Graph 1500.In addition to the MIC and HC50 values reflected in Graph 1500, Table1501 also identifies the therapeutic index (SI) of the respectivepolymers and corresponding coacervate complexes. Repetitive descriptionof like elements employed in respective embodiments is omitted for sakeof brevity.

With reference to Graph 1500 and Table 1501, Polymer E alone (not incoacervate form) has an efficacy 15.6 μg/mL MIC, which is relativelyhigher compared to PolyB, which has an MIC of only 0.5 μg/mL. Polymer Ealso has a relatively low selectivity/toxicity toward RBCs representedby an HC50 value of 500 μg/mL. However, when Polymer E is combined withPolymer A to form Complex E, the selectivity substantially increasesrelative to Polymer E alone. In particular, compared to the HC50 valueof 500 μg/mL for Polymer E, the HC50 value for Complex E skyrockets tobeyond 2500 μg/mL. This is achieved with only a minor increase in theMIC amount for Complex E relative to Polymer E (from 15.6 μg/mL forPolymer E to 31.25 μg/mL for Complex E). Thus, Complex E demonstratesextremely low toxicity toward RBCs while providing strong antimicrobialefficacy. Based on this comparison of Polymer E with Complex E, it isevident that the coacervate form significantly increases selectivitywithout diminishing the antimicrobial efficacy.

FIG. 16 presents Table 1600 illustrating characteristics of examplecoacervate Complexes HJ with varying degrees of polymerization (DP). MICvalues were obtained in K. pneumoniae (ATCC 700603). The coacervateComplexes shown in Table 1600, respectively include coacervate complexesformed with cationic Polymer H (cationic dansylated polyguanidiniumhaving chemical Formula 15), and anionic Polymer J with a DP of 10, 20and 30. Similar to the other coacervate complexes described herein,Complex HJ also demonstrates excellent physical characteristics forusage as an antimicrobial/anticancer agent. For example, with referenceto Complex HJ with a DP of 20 (e.g., n=20), the complex has a size near32 nm, a PDI of about 0.07±0.04 and a close to neutral surface (zetapotential: near −2.5±2.4 mV) when the acid/guanidinium molar ratio is1:1. Complex HJ also has comparable antimicrobial activity against K.pneumoniae as compared to the polyguanidinium when the acid block has aDP of 20 and the acid/guanidinium molar ratio is 1.1 For example, asshown in Table 1600, Complex HJ with a DP of 20 and molar ratio of 1:1has an MIC of 15.6 μg/mL which is similar to that of dansylatedguanidinium Polymer H alone, which has an MIC of 7.8 μg/mL.

FIG. 17 presents a Graph 1700 illustrating the biocompatibility/toxicityof various therapeutic polymers and coacervate complexes with mammaliancells in accordance with various embodiments described herein. Moreparticularly, Graph 1700 reflects the biocompatibility/toxicity of thevarious therapeutic polymers and coacervate complexes with THP-1 cells(a human monocytic cell line) as a function of cell viability atincrementally increased dosages. In accordance with the data reflectedin Graph 1700, different dosages (e.g., from 2 ppm to 1000 ppm in μg/mL)of the respective polymers and coacervate complexes were incubated withthe THP-1 cells for 24 hours at 37° C. The polymers tested includePolymer A, Polymer B, Polymer C, Polymer D, and Polymer E. Thecoacervates tested include Complex B, Complex C, Complex D, and ComplexE. The percent cell viability detected at concentrations less than 63ppm were substantially the same as those shown for 63 ppm, and thus arenot depicted to minimize the complexity of the visualization.

With reference to the respective bars corresponding to Polymer E andComplex E, as the dosage increased from 63 ppm to 1000 ppm, thedifference between the percent cell viability for Polymer Esignificantly drops from about 90% to about 25%. However, the percentcell viability for the coacervate formed with Polymer E and Polymer A,Complex E, remained substantially at or near 100%, event up to 1000 ppm.Similarly, with reference to the bars for Polymer D and Complex D, thepercent cell viability for Polymer D alone dropped from more than 80% at63 ppm to 5% at 1000 ppm, while Complex D demonstrated a cell viabilityat about 90% from 63 ppm to 1000 ppm. In this regard, Complex D andComplex E mitigated the toxicity of polymers D and E respectively, anddid not showed significant cytotoxicity even at concentrations of 1000ppm (in μg/mL). Thus, the disclosed coacervates, particularly Complex Eand Complex D, demonstrate strong specificity toward bacteria cells andno or low toxicity toward mammalian cells.

In contrast, FIG. 18 provides a Graph 1800 illustrating the cellviability of THP-1 with PolyB and a coacervate complex formed with PolyBand Polymer A, referred to herein as Complex H. As shown in Graph 1800,PolyB demonstrates high toxicity toward mammalian cells, with a 50% cellviability at about 250 ppm, and about a 30% cell viability at about 500ppm, dropping to below 20% at 1000 ppm. Furthermore, the integrationPolyB with Polymer A into the coacervate Complex H, did notsubstantially mitigate the toxicity of PolyB at 500 ppm or greater. Inthis regard, in contrast to Complex D and Complex E, Complex X wasunable to mitigate the toxicity of the corresponding cationicantimicrobial polymer alone high concentrations (e.g., 500 ppm orgreater).

FIG. 19 presents a graph 1900 illustrating the intracellular infectionclearance of example coacervates with P. aeruginosa in accordance withvarious embodiments described herein. Not all bacteria cells arefloating freely within an infected body. Many bacterial infectionsinvolve intracellular infections, wherein bacteria cells translocateinto other cells and macrophages. In order to combat intracellularbacterial infections, the antibacterial agent must possess an ability tokill intracellular bacteria cells.

Graph 1900 demonstrates the results of an experiment in which Complex B,Complex C, Complex D and Complex E were respectively incubated with asample comprising intracellular P. aeruginosa for a period of 1 hour. Asshown in Graph 1900, each of these respective complexes successfullyeradicated 99% of intracellular P. aeruginosa after incubation for only1 hour. Thus, as exemplified with reference to Graph 1900, Complex B,Complex C, Complex D and Complex E demonstrate strong antimicrobialefficacy against intracellular bacterial infections, including P.aeruginosa.

FIGS. 20A and 20B present graphs further illustrating the intracellularinfection clearance of Complex E with P. aeruginosa in accordance withvarious embodiments described herein. FIG. 20A presents a Graph 2001demonstrating incubation of Complex E with samples comprising cells withintracellular P. aeruginosa at different dosages of 5×MIC over a periodof 24 hours. In accordance with Graph 2001, a first sample ofintracellular P. aeruginosa was given a single dose (S.D.) of Complex Eand incubated for 24 hours. A second sample of intracellular P.aeruginosa was given double doses (D.D.) of Complex E, at 5×MIC. Inparticular, with the second sample, a single dose (S.D.) of Complex Einitially (at 0 hours) and given a second dose one hour later and leftto incubate for the remaining 23 hours. FIG. 20B presents a Graph 2002demonstrating incubation of Complex E with samples comprising cells withintracellular P. aeruginosa with double dosages of 5×, 10× and 15×MICover a period of 24 hours. In accordance with Graph 2002, for all threesamples (a first given 58 MIC, a second giving 10×MIC and a third given15×MIC), the first dose was administered at time 0 hours and the seconddose was administered two hours later.

As shown in Graphs 2001 and 2002, as the dose and MIC level increasedthe amount of eradicated intracellular P. aeruginosa also increased.This demonstrates that the dose of Complex E can be calibrated toselectively eradicate 100% of intracellular P. aeruginosa.

FIGS. 21A and 21B provides tables illustrating the in vivo toxicity andantimicrobial efficacy of coacervate complexes in accordance withvarious embodiments described herein. FIG. 21A provides a Table 2101demonstrating the in-vivo toxicity in mice, and FIG. 21B provides aTable 2102 demonstrating the in-vivo antimicrobial efficacy. The datapresented in both tables 2101 and 2102 reflect testing using a mousemodel.

With reference to FIG. 21 and Table 2101 the in vivo toxicity ofdifferent antimicrobials was measured as a function of the lethal dose50 (LD50) values (which is the amount of antimicrobial that kills 50% ofthe tested population). As shown in Table 2101, the commercialantimicrobial PolyB exhibits extremely high toxicity with an LD50 valueof 5.4 milligrams per kilogram (mg/kg). This high toxicity level doesnot change when PolyB is administered in a coacervate complex withPolymer A. Polymer E alone provides a much better toxicity levelrelative to PolyB, with an LD50 value between 17.5 and 50 mg/kg, whichis below the therapeutic does. However, this does not compare totoxicity level of Complex E. In this regard, Complex E has an LD50 valuethat goes beyond 175 mg/kg. In fact, the LD50 level of Complex E is sohigh that is cannot be measured. This demonstrates that Complex E is anextremely safe and non-toxic to mammals. This in-vivo toxicity data isfurther commensurate with the in vitro toxicity data and in vitrohemolysis data discussed above.

With reference to FIG. 21B, the data in Table 2102 reflect treatment ofa blood infected mouse model with Complex E and Complex E′. Inaccordance with the results shown in Table 2102, mice infected with 200μL of P. aeruginosa at 5 to 7×10⁶ CFU/mL, with a body weight between 23and 25 grams, were injected with either Complex E or Complex E′.Interestingly, although Complex E demonstrate impressive in vitroantibacterial efficacy, when used in vivo, Complex E′ proved to be moreeffective in vivo. In this regard, the mice treated with Complex E wereunable to survive the infection with a dose of 42.2 mg/kg. However, micetreated with Complex E′ were effectively cured with a dose of 42.2mg/kg. These results demonstrate that the addition of biotin to thedisclosed cationic polymers significantly enhances their antimicrobialefficacy of the polymer in vivo.

Thus far, the antimicrobial selectivity and efficacy of the disclosedtherapeutic coacervates has been demonstrated. In addition toantimicrobial efficacy, one or more embodiments of the disclosedcoacervates can also serve as an excellent anticancer agent againstvarious cancer cell lines with enhanced selectivity and efficacyrelative to solo cationic therapeutic polymers. FIGS. 22-24 demonstratesome example anticancer properties of various coacervates in accordancewith one or more embodiments described herein. Repetitive description oflike elements employed in respective embodiments is omitted for sake ofbrevity.

FIG. 22 provides a Graph 2200 demonstrating the efficacy of variouscoacervate complexes against BT-474 cancer cells in accordance withvarious embodiments described herein. The coacervate complexes testedinclude Complex B′, Complex C′, Complex BG′, Complex B, Complex C, andComplex BG. The anticancer efficacy of the respective coacervates wastested as a function of cell viability using an MTT assay. As shown inGraph 2200, all of the tested coacervate complexes demonstrated stronganticancer efficacy with IC50 concentrations between 63 and 125 μg/mL.The respective complexes further substantially eradicate the diseasedcells at concentrations greater than 125 μg/mL ppm. In this regard,taking into account that that the respective complexes are two componentsystems wherein 50% of the complex is a sulfonate, and only a portion ofthe cationic polymer comprises the active therapeutic component, therelative amount of active agent represented in the 63 to 125 μg/mL isless than half. Thus, the relative amount of toxic active agent includedin the dosage amount required to eradicate the cancer cells issubstantially less than 63 to 125 μg/mL. Furthermore, as demonstratedwith reference to the cytotoxicity and hemolysis data presented above,the toxicity of the example coacervates remains low even at highconcentrations (e.g., concentrations greater than a LD50 value of 175mg/kg).

FIG. 23 provides a Graph 2300 demonstrating the efficacy of coacervatecomplexes with urea against BT-474 cancer cells in accordance withvarious embodiments described herein. In particular, Graph 2300demonstrates the efficacy of Complex D and Complex D′ (whichrespectively include a small amount of urea), and Complex E and ComplexE′ (which respectively include a small amount of urea and mannose). Asshown in Graph 2300, Complex D demonstrated high efficacy against BT-474with a 50% cell viability at only 31 μg/mL and less than 20% cellviability at 63 μg/mL or greater. However, Complex D′ with the addedbiotin only demonstrated significant efficacy at much highconcentrations (e.g., 500 to 1000). This indicates that the presence ofurea negatively interacts with biotin, causing the biotin functionalgroup to be sequestered into the core of the micelle. Complex E andComplex E′ also demonstrated efficacy against BT-474, however only athigh concentrations (e.g., greater than 500 or 1000 ppm). Accordingly,Complex D is the more promising anticancer agent against BT-474.

FIG. 24 presents a Chart 2400 illustrating the cytotoxicity of acoacervate Complex D against healthy non-cancerous mammalian cell lineHEK293 in accordance with various embodiments described herein. Inaccordance with Chart 2400, the cytotoxicity of Complex D against HEK293was tested as a function of polymer concentration using an MTT assay. Asshown in Chart 2400, almost 100% of the HEK293 cells survived atconcentrations of Complex D at the therapeutic amount of 31 ppm (μg/ml).This high cell viability remained even up to concentrations up to 500ppm. Complex D further demonstrated an IC₅₀ of 1000 μg/mL and thus aselectivity of=1000/31=32. This demonstrates that Complex D not onlydemonstrates strong efficacy as an anticancer agent against BT-474, butalso demonstrate strong selectivity toward diseased cells over healthymammalian cells, and those poses a very low toxicity level.

FIG. 25 presents a high-level flow diagram of an example method 2500 forenhancing the selectivity and efficacy of therapeutic polymers against abroad spectrum of pathogens and cancer cell lines using a coacervatecomplex in accordance with various embodiments described herein.Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

At 2502, a cationic therapeutic polymer (e.g., Polymer E, Polymer B,Polymer C, Polymer F, Polymer D, Polymer G, BG and the like) can bemixed with an anionic polymer (e.g., Polymer A, Polymer A′ and the like)in solution (e.g., water, serum, etc.). At 2504, a coacervate complexcan be formed between the cationic therapeutic polymer and the anionicpolymer as a result of the mixing, wherein the coacervate providesantimicrobial functionality or anticancer functionality.

FIG. 26 presents a high-level flow diagram of an example method 2600 forenhancing the selectivity and efficacy of therapeutic polymers against abroad spectrum of pathogens and cancer cell lines using a coacervatecomplex comprising a biotinylated anionic polymer in accordance withvarious embodiments described herein. Repetitive description of likeelements employed in respective embodiments is omitted for sake ofbrevity.

At 2602 an anionic polymer can be formed comprising a biotin functionalgroup bound to an end of a molecular backbone of the anionic polymer(e.g., Polymer A′ or the like). At 2604, a cationic therapeutic polymer(e.g., Polymer E, Polymer B, Polymer C, Polymer F, Polymer D, Polymer G,and the like) can be mixed with an anionic polymer in solution, whereinbased on the mixing, the anionic polymer and the cationic therapeuticpolymer form a coacervate complex that provides one or more therapeuticfunctionalities selected from a group consisting of an antimicrobialfunctionality and an anticancer functionality

III—Fluorescence Coacervate Diagnostics

One or more additional embodiments of the disclosed subject matter aredirected to usage of the above described coacervates for diagnosticpurposes. In accordance with these embodiments, a fluorescent dyefunctional group can be attached to the aniconic polymer and combinedwith a cationic polymer to form a coacervate complex in solution (e.g.,DI water). The cationic polymer can be calibrated to target or reactwith a specific pathogen and/or cancer cell type. The fluorescentproperties of the dye are quenched as a result of the formation of thecoacervate complex when the functionalized anionic polymer is combinedwith the cationic polymer. However, when the coacervate opens up as aresult of interaction between the cationic polymer with the targetpathogen or cancer cell type, the anionic polymer is released and thefluorescent dye illuminates. Thus, detection of luminescence in responseto exposure of the cationic complex to a fluid sample (e.g., urine,saliva, etc.) can indicate the specific target pathogen or cancer cellis present.

FIG. 27 provides a diagram illustrating a fluorescence based coacervatediagnostic process 2700 in accordance with various embodiments describedherein. Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

In accordance with process 2700, an anionic polymer such as Polymer A,Polymer A′ or the like, can be labeled with a fluorescent die to formfluorescent anionic polymer 2702 comprising fluorescent functionalgroups 2704. The fluorescent anionic polymer 2702 can further becombined with selective cationic polymer 2706 that has been calibratedor tailored to only react with a specific target biomarker, such as aspecific type of bacteria and/or bacteria strain, a specific type ofcancer cell, or the like. For example, in various embodiments, theselective cationic polymer 2706 can comprises a functional guanidiniumcationic homopolymer (e.g., Polymer B, Polymer C, Polymer BG, etc.), afunctional guanidinium cationic copolymer (e.g., Polymer D, Polymer E,etc.), a block copolymer or the like. When the fluorescent anionicpolymer 2702 is separated from the selective cationic polymer 2707, thefluorescent functional groups 2704 can be configured to illuminate.However, when the fluorescent anionic polymer 2702 and the selectivecationic polymer 2706 self-assemble into a coacervate complex 2708, thefluorescent functional groups 2704 become quenched and an unable to emitlight (as indicated by the change from the fluorescent functional groups2704 to the quenched state 2704′).

This quenched form of coacervate complex 2708 can further be mixed witha clinical sample of comprising biological fluid (e.g., urine, saliva,interocular fluid, blood, etc.) to facilitate detecting presence of thetarget biomarker. In this regard, if the biomarker (e.g., the specifictarget bacteria type/strain, the specific cancer cell type, etc.) ispresent in the clinical sample, the selective cationic polymer withinteract with the target biomarker, resulting in the release of thefluorescent anionic polymer 2702 therefrom and the unquenching of thefluorescent functional groups. As a result, the functional groups willemit light. For example, in various embodiments, the selective cationicpolymer 2706 can interact with the anionic surface of the bacterialmembrane 2710 (or the cancer cell membrane) and perform an ion exchangetherewith, causing the fluorescent anionic polymer 2702 to be released.In this regard, the presence and/or amount of the target biomarkerpresent in the clinical sample can be determined based on detection oflight/photon emission and/or an amount of light/photons emitted from theclinical sample. In various embodiments, a fluorometer or anothersuitable instrument can be used to detect and measure the fluorescentlight/photon emission. The amount of light/photons emitted can thus becorrelated to a specific target biomarker which can further becorrelated to a specific infection, disease, condition, etc.

FIG. 28 presents a Graph 2800 illustrating the fluorescence quenchingcharacteristics of diagnostic coacervate complexes in accordance withvarious embodiments described herein. As shown in Graph 2800, the freefloating, fluorescent anionic polymer 2702 emits a significantly higherwavelength relative to the quenched coacervate complexes.

FIG. 29 presents a high-level flow diagram of an example method 2900 fordiagnosing a disease or condition using a fluorescence based coacervateassay in accordance with various embodiments described herein.Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

At 2902, an anionic polymer can be formed comprising a fluorescentfunctional group bound to an end of a molecular backbone of the anionicpolymer. At 2904, the anionic polymer can be mixed with cationic polymerin solution, wherein based on the mixing, the anionic polymer andcationic polymer form a coacervate complex that quenches light emissionof the fluorescent functional group, and wherein the cationic polymerreacts with a known biomarker. At 2906, the coacervate complex can bemixed with a biological fluid sample, and at 2908, presence of the knownbiomarker can be detected in the biological fluid sample based on anamount of the light emission detected after the mixing of the coacervatecomplex with the biological fluid sample.

What has been described above includes examples of the embodiments ofthe present invention. It is, of course, not possible to describe everyconceivable combination of components or methodologies for purposes ofdescribing the claimed subject matter, but it is to be appreciated thatmany further combinations and permutations of the subject innovation arepossible. Accordingly, the claimed subject matter is intended to embraceall such alterations, modifications, and variations that fall within thespirit and scope of the appended claims. Moreover, the above descriptionof illustrated embodiments of the subject disclosure, including what isdescribed in the Abstract, is not intended to be exhaustive or to limitthe disclosed embodiments to the precise forms disclosed. While specificembodiments and examples are described in this disclosure forillustrative purposes, various modifications are possible that areconsidered within the scope of such embodiments and examples, as thoseskilled in the relevant art can recognize.

In this regard, with respect to any figure or numerical range for agiven characteristic, a figure or a parameter from one range may becombined with another figure or a parameter from a different range forthe same characteristic to generate a numerical range. Other than in theoperating examples, or where otherwise indicated, all numbers, valuesand/or expressions referring to quantities of ingredients, reactionconditions, etc., used in the specification and claims are to beunderstood as modified in all instances by the term “about.”

While there has been illustrated and described what are presentlyconsidered to be example features, it will be understood by thoseskilled in the art that various other modifications may be made, andequivalents may be substituted, without departing from claimed subjectmatter. Additionally, many modifications may be made to adapt aparticular situation to the teachings of claimed subject matter withoutdeparting from the central concept described herein. Therefore, it isintended that claimed subject matter not be limited to the particularexamples disclosed, but that such claimed subject matter may alsoinclude all aspects falling within the scope of appended claims, andequivalents thereof.

In addition, while a particular feature of the subject innovation mayhave been disclosed with respect to only one of several implementations,such feature may be combined with one or more other features of theother implementations as may be desired and advantageous for any givenor particular application. Furthermore, to the extent that the terms“includes,” “including,” “has,” “contains,” variants thereof, and othersimilar words are used in either the detailed description or the claims,these terms are intended to be inclusive in a manner similar to the term“comprising” as an open transition word without precluding anyadditional or other elements.

Moreover, the words “example” or “exemplary” are used in this disclosureto mean serving as an example, instance, or illustration. Any aspect ordesign described in this disclosure as “exemplary” is not necessarily tobe construed as preferred or advantageous over other aspects or designs.Rather, use of the words “example” or “exemplary” is intended to presentconcepts in a concrete fashion. As used in this application, the term“or” is intended to mean an inclusive “or” rather than an exclusive“or”. That is, unless specified otherwise, or clear from context, “Xemploys A or B” is intended to mean any of the natural inclusivepermutations. That is, if X employs A; X employs B; or X employs both Aand B, then “X employs A or B” is satisfied under any of the foregoinginstances. In addition, the articles “a” and “an” as used in thisapplication and the appended claims should generally be construed tomean “one or more” unless specified otherwise or clear from context tobe directed to a singular form.

What is claimed is:
 1. A method, comprising: forming a therapeuticpolymer based on polymerization of a plurality of therapeutic monomers,wherein the therapeutic polymer provides a therapeutic functionality;and attaching biotin to the therapeutic polymer, resulting in abiotin-functionalized therapeutic polymer, wherein thebiotin-functionalized therapeutic polymer provides greater therapeuticefficacy relative to the therapeutic polymer.
 2. The method of claim 1,wherein the biotin-functionalized therapeutic polymer provides thegreater therapeutic efficacy based on increased uptake of thebiotin-functionalized therapeutic polymer by pathogens or cancer cellsrelative to the therapeutic polymer.
 3. The method of claim 1, whereinthe biotin-functionalized therapeutic polymer provides the greatertherapeutic efficacy based on reduced toxicity of the therapeuticpolymer toward mammalian cells relative to the therapeutic polymer. 4.The method of claim 1, wherein the therapeutic functionality comprisesan anticancer functionality.
 5. The method of claim 1, wherein thetherapeutic functionality comprises an antimicrobial functionality. 6.The method of claim 1, wherein the therapeutic polymer comprisespolyguanidinium.
 7. The method of claim 1, wherein the attachingcomprises performing the polymerization of the plurality of therapeuticmonomers in presence of biotinol, resulting in formation of thebiotin-functionalized therapeutic polymer with the biotin bound to anend of the polymer backbone.
 8. The method of claim 7, wherein thepolymerization comprises a ring-opening polymerization.
 9. The method ofclaim 1, wherein the attaching comprises attaching the biotin after thepolymerization.
 10. A therapeutic polymer, comprising: a polymerbackbone; therapeutic functional groups bound to the polymer backbone;and a biotin-based functional group bound to an end of the polymerbackbone.
 11. The therapeutic polymer of claim 10, wherein thetherapeutic functional groups comprise guanidinium moieties.
 12. Thetherapeutic polymer of claim 10, wherein the therapeutic polymerfacilitates necrosis of bacteria cells.
 13. The therapeutic polymer ofclaim 10, wherein the therapeutic polymer facilitates autophagy ofcancer cells.
 14. The therapeutic polymer of claim 10, wherein thebiotin-based functional group comprises biotinol.
 15. The therapeuticpolymer of claim 10, wherein the therapeutic polymer has a chemicalstructure characterized by Formula I:

wherein n represents an integer between 10 and 50, wherein R₁ comprisesthe biotin-based functional group, and wherein R₂ comprises a spacergroup.
 16. The therapeutic polymer of claim 10, wherein the therapeuticpolymer has a chemical structure characterized by Formula II:

wherein n represents an integer between 10 and
 50. 17. The therapeuticpolymer of claim 10, wherein the therapeutic polymer has a chemicalstructure characterized by Formula III:

wherein n represents an integer between 10 and 50 wherein m representsan integer between 1.0 and 10, and wherein R₁ comprises the biotin-basedfunctional group.
 18. The therapeutic polymer of claim 10, wherein thetherapeutic polymer has a chemical structure characterized by FormulaIV:

wherein n represents an integer between 10 and 50, wherein R₁ comprisesa functional group, wherein R₂ comprises the biotin-based functionalgroup.
 19. A therapeutic polymer, wherein the therapeutic polymer has achemical structure characterized by Formula I:

wherein n represents an integer between 10 and 50, wherein R₁ comprisesthe biotin-based functional group, and wherein R₂ comprises a spacergroup.
 20. The therapeutic polymer of claim 19, wherein the therapeuticpolymer is an anticancer agent.
 21. The therapeutic polymer of claim 19,wherein the therapeutic polymer is an antimicrobial agent.
 22. Ananticancer agent, wherein the anticancer agent has a chemical structurecharacterized by Formula I:

wherein n represents an integer between 10 and 50, and where theanticancer agent is effective against a plurality of different cancercell lines.
 23. The anticancer agent of claim 22, wherein the pluralityof different cancer cell lines comprises cancer cell line BT-474.
 24. Amethod, comprising: polymerizing guanidinium-functionalized cycliccarbonate monomers via a ring opening polymerization reaction usingbiotinol as an initiator; and forming a biotinylated polyguanidiniummacromolecule based on the polymerizing.
 25. The method of claim 24,wherein the biotinylated polyguanidinium provides antimicrobial andanticancer functionality.